cAMP has been suggested to play two important roles in yeast: a role as a signal for specific protein turnover during catabolite inactivation and a role in cell division and differentiation. Our overall aim is to study the mechanisms of how cAMP influences these two processes. Catabolite inactivation is the glucose induced, rapid and selective degradation of certain proteins in yeast. Studies on the catabolite inactivation of fructose 1,6-bisphosphatase (FBPase) in Saccharomyces cerevisiae have suggested that the cAMP-dependent phosphorylation of FBPase as the targeting event which marked the enzyme for degradation. Our studies on FBPase and cytoplasmic malate dehydrogenase (cMDH) showed that the two enzymes are catabolite inactivated by two different mechanisms. No in vitro phosphorylation of cMDH was detected. Catabolite inactivation of FBPase depends on vacuoles while cMDH inactivation does not. The relevance of cAMP in catabolite inactivation will be studied using various adenylate cyclase and cAMP-dependent protein kinase mutants. Catabolite inactivation will be followed by immunoblotting technique, by measurements of specific activities and will be correlated with cAMP concentrations and the degree of protein phosphorylation. Proteolytic succeptability of phosphorylated and unphosphorylated FBPase will be compared. In vitro inactivation systems will be developed and the involvement of vacuoles in the proteolysis of FBPase will be studied. Involvement of ubiquitin conjugation system on cMDH proteolysis will be examined. Involvement of ubiquitin conjugation system on cMDH proteolysis will be examined. We have found a protein which cross-react immunologically with the regulatory (R) subunit of cAMP-dependent protein kinase but has a higher Mr than R subunit. The regulatory subunit-related protein will be purified and characterized. The fatty acid moiety of yeast RAS proteins will be determined. The enzyme system catalyzing the addition of fatty acid to RAS proteins will be studied using peptide analog as substrate. Several proteins are highly phosphorylated when yeast cell division is arrested at G1 "start" due to either the lowering of cAMP content in cAMP-requiring (cyr) cells or the shifting of cdc35 and cdc25 to the non-permissive temperature. The most prominent of these proteins has an Mr=72K (pp72) and this protein is also found to be highly phosphorylated when wild type cells enter into stationary phase. We will purified and characterized pp72 biochemicaly and cytochemically.